Enhanced detection of CNS cell secretome in plasma protein-depleted cerebrospinal fluid

Human cerebrospinal fluid (CSF) proteome is actively investigated to identify relevant biomarkers and therapeutic targets for neurological disorders. Approximately 80% of CSF proteome originate from plasma, yielding a high dynamic range in CSF protein concentration and precluding identification of potential biomarkers originating from CNS cells. Here, we have adapted the most complete multiaffinity depletion method available to remove 20 abundant plasma proteins from a CSF pool originating from patients with various cognitive disorders. We identified 622 unique CSF proteins in immunodepleted plus retained fractions versus 299 in native CSF, including 22 proteins hitherto not identified in CSF. Parallel analysis of neuronal secretome identified 34 major proteins secreted by cultured cortical neurons (cell adhesion molecules, proteins involved in neurite outgrowth and axonal guidance, modulators of synaptic transmission, proteases and protease inhibitors) of which 76% were detected with a high confidence in immunodepleted CSF versus 50% in native CSF. Moreover, a majority of proteins previously identified as secretory products of choroid plexus cells or astrocytes were detected in immunodepleted CSF. Hence, removal of 20 major plasma proteins from CSF improves detection of brain cell-derived proteins in CSF and should facilitate identification of relevant biomarkers in CSF proteome profiling analyses.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long‐chain base intermediates. The formation and function of ceramide phosphates (Cer‐Ps) in chilled Arabidopsis were explored. Cer‐Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold‐stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer‐P occurs in cold‐stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer‐P formation is independent of long‐chain base phosphate (LCB‐P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA‐dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer‐P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.     

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.     

Association of macro-level determinants with adolescent overweight and suicidal ideation with planning: A cross-sectional study of 21 Latin American and Caribbean Countries

BackgroundAdolescents and young people (10–24 years old) in the Latin America and the Caribbean (LAC) region represent approximately 25% of the region’s population. Since the 2008 global economic crisis, the pace of reduction in poverty and income inequality in the LAC region has stalled. The region is characterised by high levels of inequities and is also vulnerable to many natural disasters. Food systems are changing with increased availability and marketing of packaged and fast foods and sugar-sweetened drinks. Adolescence is a formative phase of the life course with multiple physical, emotional and social changes which can make them vulnerable to health problems. We assess the potential impact of macro-determinants, human and economic development as well as income inequality, on 2 top-ranking regional priorities for adolescent nutrition and mental health, using measures of overweight and suicidal ideation and planning which some have shown to be associated.Methods and findingsThe Global School-based Health Survey (GSHS) is a nationally representative self-administered, school-based survey. We examined overweight/obesity and suicidal ideation with planning by gross domestic product (GDP) per capita or human development index (HDI) in 10–19-year-old adolescents from 21 LAC countries between 2009 and 2013. Sample sizes varied from 943 in Anguilla to 27,988 in Argentina. A total of 55,295 adolescents had a measure of overweight/obesity status, and 59,061 adolescents reported about suicidal ideation with planning. There was equal representation by sex in the surveys (52% girls and 48% boys). A total of 28.8% of boys and 28.1% of girls had overweight/obesity, and 7.5% of boys and 17.5% of girls reported suicidal ideation with planning over the last 12 months. Adjusted for individual socioeconomic and risk behaviours, and relative to the highest GDP per capita tertile, the middle tertile was associated with 42% (95% confidence interval (CI) 59% to 17%, p = 0.003) and 32% (95% CI 60% to 5%, p = 0.023), and the lowest tertile with 40% (95% CI 55% to 19%, p = 0.001) and 46% (95% CI 59% to 29%, p < 0.001) lower chances of overweight/obesity for girls and boys, respectively. A similar positive effect was seen with HDI, with lowest chances of overweight in the lowest tertile compared with the highest tertile for both sexes. Overweight/obesity was positively related with suicidal ideation with planning for girls (odds ratio (OR) 1.12, 95% CI 1.02 to 1.22, p = 0.009) and weakly related for boys (OR 1.09, 95% CI 0.96 to 1.24, p = 0.182). In contrast to overweight/obesity status, suicidal ideation with planning was not related to macro-level indices despite both outcomes sharing common individual socioeconomic and risk behaviour correlates. Limitations include the dominance of Argentinians in the sample (40%), the exclusion of vulnerable adolescents who dropped out of school, and reporting bias due to stigma of mental health–related issues.ConclusionsThis study shows that economic and human development were positively associated with adolescent overweight/obesity but not with suicidal ideation with planning. We also observed an interconnectedness between overweight/obesity and suicide ideation with planning among girls. These findings highlight the importance of strategies that engage with both upstream and downstream determinants to improve adolescent nutrition and mental health.     

Lipogenesis in the liver and adipose tissue of the cardiomyopathic hamster.

The activities of fatty acid synthetase, citrate cleavage enzyme, glucose-6-phosphate dehydrogenase and malic enzyme were measured in the liver and adipose tissue of cardiomyopathic and normal hamsters at age 33, 68 and 108 days. There was no difference in the activity of hepatic fatty acid synthetase between the diseased animals and the controls at any stage in their development. The activity of glucose-6-phosphate dehydrogenase was not different until age 108 days where it was significantly elevated in the BIO 82.62 strain. Citrate cleavage enzyme in the liver was depressed at all stages in the diseased animals as was malic enzyme. In adipose tissue, all enzyme activities were significantly depressed in the cardiomyopathic animals at the three stages. These data suggest that lipogenesis was depressed in the cardiomyopathic hamster.     

Functional analysis of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> subpopulations associated with artemisinin resistance in Cambodia

Background

Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008–2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs.

Results

Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population.

Conclusions

Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations.

     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.